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Aims: To investigate the expression of pepsinogen A3 (Pg3) encoding genes in the gastric mucosa of normal controls and subjects with atrophic gastritis and gastric cancer., Methods: One hundred and fifty nine patients underwent upper gastrointestinal endoscopy with sampling of gastric biopsy specimens and serum. Pg3 isoproteins were determined by electrophoresis in serum and gastric mucosal biopsy specimens. Pg3 encoding genes were assessed by PCR in DNA obtained from peripheral blood., Results: One hundred and one subjects (82 normal histology/chronic gastritis, 17 atrophic gastritis, two gastric cancer) showed a pepsinogen phenotype with presence of Pg3 and a corresponding pepsinogen genotype with presence of Pg3 encoding genes. Fifty eight subjects showed a phenotype lacking Pg3. In 39 of them (23 normal histology/chronic gastritis, 11 atrophic gastritis, five gastric cancer), a corresponding genotype without Pg3 encoding genes was found. However, in the remaining 19 subjects (4 normal histology/chronic gastritis, nine atrophic gastritis, six gastric cancer); Pg3 encoding genes were demonstrable in the absence of Pg3 production., Conclusions: Unexpressed Pg3 encoding genes can be shown in many cases of atrophic gastritis and gastric cancer, but rarely in healthy controls and subjects with superficial gastritis. The correlation of atrophic gastritis and gastric cancer with a pepsinogen phenotype lacking Pg3 can be explained by loss of expression of Pg3 encoding genes throughout the complete gastric mucosa. The mechanism of such loss and the importance as a marker for premalignant degeneration have to be elucidated.

Two pepsinogens (pepsinogens 1 and 2) were purified from the esophageal mucosa of the bullfrog (Rana catesbeiana), and their molecular weights were determined to be 40,100 and 39,200, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal 70-residue sequences of both pepsinogens are the same, including the 36-residue activation segment. Furthermore, a cDNA clone encoding frog pepsinogen was obtained and sequenced, which permitted deduction of the complete amino acid sequence (368 residues) of one of the pepsinogen isozymogens. The calculated molecular weight of the protein (40,034) coincided well with the values obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results are incompatible with the previous report (Shugerman R. P., Hirschowitz, B. I., Bhown, A. S., Schrohenloher, R. E., and Spenney, J. G. (1982) J. Biol. Chem. 257, 795-798) that the major pepsinogen isolated from the bullfrog esophageal gland is a unique "mini" pepsinogen with a molecular weight of approximately 32,000-34,000. The two pepsinogens were immunologically indistinguishable from each other and related to human pepsinogen C. The deduced amino acid sequence was also more homologous with those of pepsinogens C than those of pepsinogens A and prochymosin. These results indicate that the frog pepsinogens belong to the pepsinogen C group. They were both glycoproteins, and therefore, this is the first finding of carbohydrate-containing pepsinogens C. Both pepsinogens were activated to pepsins in the same manner by an apparent one-step mechanism. The resulting pepsins were enzymatically indistinguishable from each other, and their properties resembled those of tuna pepsins.

In order to clarify the structure and development of rabbit pepsinogens, purification and molecular cloning of these proteins were performed at various developmental stages. Several pepsinogens were isolated, and they were classified as pepsinogens F and M, and into pepsinogen groups I, II, and III. The relative levels and specific activities of the various pepsinogens changed significantly during development. Pepsinogens F and M were present only at the early postnatal stage, and their level was higher than those of other pepsinogens at this stage. Pepsinogens in groups I, II, and III were the predominant zymogens at the late postnatal stage. cDNA clones encoding all of these pepsinogens were obtained, with the exception of pepsinogens I and M, and the nucleotide sequences were determined. Each cDNA contained a leader region (signal peptide), a pro-region (activation segment), and a pepsin region, of 15, 44, and 328 residues, respectively, with the exception of the cDNA for pepsinogen F in which the pro- and pepsin regions were composed of 43 and 330 residues, respectively. Pepsinogens in groups II and III exhibited a high degree of similarity with one another, whereas many substitutions were found in pepsinogen F. A unique substitution in the activation segment of pepsinogen F, namely, Gly----Asp at position 21, was found, which made the structural features of this segment more specific. A phylogenic tree was constructed from the differences in nucleotide sequences and showed clearly that each pepsinogen in groups II and III could be classified as pepsinogen A, a major pepsinogen in mammals. Pepsinogen F diverged significantly from these groups and may be a new type of pepsinogen. Northern analysis revealed that the expression of the gene for pepsinogen F was restricted to the early postnatal stage, and the expression of genes for pepsinogens in groups II and III was detected predominantly at later stages, a result that shows the switching of gene expression from fetal pepsinogen to adult pepsinogens during development.

Pepsinogen A (PGA) isozymogen patterns in urine and gastric mucosa can be visualised in non-denatured polyacrylamide gel electrophoresis by showing proteolytic activity after the conversion of pepsinogen into pepsin by acid. This method is not suitable for visualising PGA patterns in serum due to low PGA concentrations. To obtain a more sensitive visualisation method an immunoblotting technique was developed. PGA isozymogen patterns from urine and sonified gastric mucosa specimens obtained by immunoblotting were identical with those obtained by activity staining. The immunostaining method was at least 50 times more sensitive. PGA isozymogen patterns could be visualised in serum. Preliminary results suggest that the PGA patterns in serum and gastric mucosa are identical. As an association has been found between the genetically determined PGA isozymogen patterns in gastric mucosa and gastric malignancies in man, immunoblotting of PGA isozymogens in serum may provide a screening tool for subjects at risk of malignant gastric disease.

Total human pepsinogen (PG) was isolated from gastric fundic mucosa and PGA (formerly called PGI) from urine, using standard ion-exchange and gel filtration techniques. Gastric PGA was separated from PGC (formerly called PGII) either by immunoaffinity or high resolution ion-exchange chromatography (fast protein liquid chromatography, Pharmacia, Uppsala, Sweden). The individual PGA isozymogens 2, 3, 4 and 5 could be isolated to homogeneity with the aid of the same ion-exchanger. Evidence was obtained for the existence of secondary modifications of the PGA fractions 3, 4 and 5, electrophoretically overlapping the primary (genetic) isozymogens.

A genetic analysis of human urinary pepsinogen isozymes is presented. Nine discrete phenotypes were identified in a population survey of 215 unrelated Caucasian individuals. The phenotypes were characterized by differences among the staining intensities of the activated group I pepsinogens, Pg 5, Pg 4, Pg 3, and Pg 2. The genetic studies demonstrated that the codominant expression of four alleles, PgA, PgB, PgC and PgD, at a single genetic locus determined the nine phenotypes identified. Linkage analysis excluded close linkage of the Pg locus with the chromosome 6 markers HLA, GLO1, and Bf.

The isozymogens PGA-3 and PGA-5 of human pepsinogen A were digested with endoproteinase Lys-C. The peptides were separated by reverse-phase HPLC. PGA-5 showed a peak strongly absorbing at 254 nm absent in PGA-3. Analysis of amino acid composition using the Pico-Tag methodology combined with DABITC-sequencing reveals the sequence Tyr-Phe-Pro-Gln-Trp-Lys (peptide 37-43 of the activation segment). This confirms a study at the DNA level by our group [16] suggesting a Glu greater than Lys mutation at position 43 in the activation segment of PGA-5. Furthermore, it is proposed that the number of genetic variants of PGA is higher than is actually seen by electrophoresis.